The long term goal of this proposal is to develop therapeutic interventions in the generation of neurons from neural stem cells (NSC) for replacement therapies of disease and injury in the central nervous system (CNS). Neural stem cells give rise to all of the different cell types in the CNS. Understanding of the complex transcriptional networks that control the process of cell fate determination in NSC is crucial in applications for therapeutic purpose. The generation of diversity of neurons and glial cells is achieved through cell proliferation, cell fate determination and differentiation of embryonic NSC populations into progressively more specialized cell types that make up the CNS. The uniqueness of individual neurons and glial cells is determined by combinatorial patterns of gene expression;and gene expression is largely controlled at the level of DNA sequence (cis-regulatory element, genetic), as well as by chromatin structure (epigenetic). One of the key components in transcription regulation is the enhancer, a non-coding DNA sequence that is often evolutionarily conserved. Upon binding of trans-acting factors, enhancers determine tissue or cell type-specific expression of particular genes. We will choose genes that are crucial to NSC cell fate determination from genome wide gene expression studies and analyze the non-coding DNA regions for their regulatory functions (e.g. as an enhancer) in NSC gene expression during cell fate determination. We will predict (in silico) and functionally characterize (in vivo) the predicted putative enhancers in chick retinal stem cells. We refer to this kind of enhancer as NSC enhancers. Our focus will be on NSC enhancers that are important for the development of the retina. The two specific aims are: 1) to predict evolutionarily conserved NSC enhancers;and 2) to verify and characterize putative NSC enhancers. The successful completion of the proposed study will help to identify transcriptional control networks that are crucial for cell fate determination of neural stem cells. In addition, novel retina-specific enhancers identified from this study can be used to identify novel protein factors that are previously unknown for their function in controlling neural cell fate determination. Our findings will ultimately provide an integrated transcription network that controls NSC cell fate determination in the retina (can also be applied to other developmental systems in general). Such an understanding of the transcriptional networks is fundamental to the development of potential treatments or therapeutic transplants for diseases ranging from retinal degeneration to spinal cord injury. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to search for DNA sequence elements that exist in the non-protein coding regions of the genome that regulate the generation of nerve cells from neural stem cells. The successful completion of the proposed studies will help the development of potential treatments or therapeutic transplants for diseases ranging from neural degeneration to spinal cord injury.